Comparison of histochemical and biochemical assays for estrogen receptor in human breast cancer cell lines.

Two human breast cancer lines, MCF-7 and T47D cells, were investigated for the presence of estrogen receptor (ER) by biochemical and histochemical techniques. Using the dextran-coated charcoal technique and isoelectric focusing, MCF-7 cells were ER positive, and T47D cells were ER negative. Fluorescein conjugates to 17 beta-estradiol by the sixth carbon (17 beta-estradiol-6-carboxymethyloxime:bovine serum albumin: fluorescein isothiocyanate and 17 beta-estradiol-6-iminooxyacetylfluoresceinamine) and by the 17th carbon [N-fluoresceino-N'-[17 beta-(estradiol hemisuccinamide)ethyl]thiourea, 17-FE] were prepared for cytochemical evaluation of the ER status of the two cell lines. The binding affinity of the estradiol conjugates for ER varied, the 17-FE conjugate having the highest affinity of 0.08 relative to 17 beta-estradiol. Following incubation with 10 nM 17-FE, both MCF-7 and T47D cells displayed cytoplasmic and nuclear fluorescent staining. Isoelectric focusing of MCF-7 cytosol incubated in the presence of 10 nM 17-FE revealed binding of the fluorescein conjugate to a protein species which did not bind 17 beta-[3H]-estradiol. Isoelectric focusing of T47D cytosol revealed binding of 17-FE to two protein components, neither one of which showed specific binding of 17 beta-[3H]estradiol. The results suggest different protein binding species for fluoresceinated estradiol conjugates and [3H]estradiol and help to explain reported differences in histochemical and biochemical ER analyses.

Natural killer cell infection and inactivation in vitro by the human immunodeficiency virus.

Cytolytic activity of human mononuclear peripheral blood leukocytes from healthy donors, cultured in interleukin-2 conditioned medium, was abrogated by in vitro infection with the lymphadenopathy associated virus (LAV) isolate of the human immunodeficiency virus (HIV). Although viral antigens are not expressed in cultured cells until 14 days postinfection, cytolytic activity was lost as early as 3 days after infection. Loss of cytolytic function was not a result of the release of suppressive factors from either infected cells or uninfected CEM cells since supernatants from neither infected cultures nor CEM cell cultures had any inhibitory effects on the function of uninfected cells. Cultured lymphocytes expressing Leu 11b were also shown to express HIV antigens via immunofluorescence after 14 days in culture. These results suggest that natural killer (NK) cells, as defined by expression of Leu 11b, were infected by HIV in vitro and the loss of lytic function was likely a direct consequence of that infection.

Molecular and morphological evidence for type B retrovirus (oncornavirus) expression in human mammary carcinoma. An overview using scanning electron microscopy, immunoperoxidase staining, and transmission electron microscopy.

Approximately 45% of human mammary carcinoma cases express an antigen which cross reacts in formalin fixed tissues with antiserum prepared against the purified 52,000 molecular weight structural glycoprotein (gp52) of mouse mammary tumor virus (MMTV). Breast carcinoma immunoperoxidase marking is abolished by antiserum adsorption with MMTV. Adsorption with murine leukemia virus failed to block immunoperoxidase marking. No correlation was seen in the cases analyzed between gp52-like antigen expression and family history of mammary carcinoma, age, or pathological classification. The evidence linking an oncornaviral agent in human mammary carcinoma is reviewed with respect to the structure and biology of a known etiological agent, MMTV, in murine mammary cancer. The potential role of SEM in amplifying the surface area available for analysis in malignant and premalignant human breast epithelia is considered.

Evaluation of antiviral drugs and neutralizing antibodies to human immunodeficiency virus by a rapid and sensitive microtiter infection assay.

A 96-well microtiter infection assay for the human immunodeficiency virus (HIV) is described. The assay utilizes human T-cell lymphotropic virus type I-immortalized MT-2 cells as targets for infection and requires only 4 to 5 days for completion. Cytolysis was quantitated by vital dye uptake of poly-L-lysine-adhered cells as an endpoint for infection. The assay's efficacy was proven by the sensitive and accurate assessment of several known anti-HIV agents including two inhibitors of reverse transcription (3'-azido-3'-deoxythymidine and 2',3'-dideoxycytidine), three biological response modifiers (recombinant interferons alpha and beta and mismatched double-stranded RNA), a direct inactivator of HIV virions (amphotericin B), and neutralizing antibodies from two HIV-positive human subjects. Evaluation of data was facilitated by computer-assisted analysis. This assay provides a means for rapid, sensitive, and inexpensive large-scale in vitro testing of potential anti-HIV therapeutic regimens and quantitation of HIV-neutralizing antibody titers.

beta-Adrenergic induced synthesis and secretion of phosphatidylcholine by isolated pulmonary alveolar type II cells.

Rat pulmonary alveolar type II cells isolated by trypsinization and discontinuous density gradient ultracentrifugation were maintained in primary culture for 48 hours. The cultured type II cells responded to beta-adrenergic, but not cholinergic, agonists by an increase in the rate of synthesis and also secretion of 3H-phosphatidylcholine. The beta-adrenergic agonists, isoproterenol and terbutaline, 10 microM, caused a 1.7-fold increase in the rate of synthesis of 3H-phosphatidylcholine after a 4-hour incubation. At this time, there was also an increase in the cAMP content of the cultured cells. Terbutaline, 10 microM, caused a 4.9-fold increase in the rate of secretion of 3H-phosphatidylcholine after a 1-hour incubation. The beta-adrenergic effect on both synthesis and secretion by type II cells was blocked by propranolol. 8-Br-cAMP, 100 microM, but not 8-Br-cGMP, mimicked the beta-adrenergic effect on both synthesis and secretion of 3H-phosphatidylcholine. The increased rate of 3H-phosphatidylcholine induced by beta-adrenergic agonists was unaffected by colchicine. These data are consistent with the hypothesis that both synthesis and secretion of pulmonary surfactant are under adrenergic control operating through a beta-receptor and the adenylate cyclase system. These data also suggest that synthesis and secretion of pulmonary surfactant are independent processes. The possibility of other neural or hormonal mechanisms is not excluded.

Inhibition of murine interferon production following in vivo administration of benzo[a]pyrene.

To investigate whether alteration in interferon (IFN) production might serve as a biomarker for certain toxic chemical exposures, an in vivo mouse model system was studied. Female C3H mice were injected intraperitoneally with varying doses of benzo[a]pyrene (BP), and at various times subsequent to this treatment, serum IFN levels, following Sendai virus induction, were determined by a cytopathic effect inhibition assay. Doses of 4.6 mg/mouse (180 mg/kg body weight) caused a significant depression in IFN production at 12, 48, and 120 h after BP administration. Doses of 0.46 mg also produced significant decreases at 48 h following exposure. At 48 h post-BP treatment, the reduction in serum IFN titers in treated animals relative to controls was: 62% for the 0.46-mg dose, and 94% for the 4.6-mg dose. These results indicate that systemic administration of BP can significantly depress the whole-animal IFN response to viral stimulation, and that such depression can persist for a rather extended period at certain dose levels.

Differential inhibition of HIV-1 cell binding and HIV-1-induced syncytium formation by low molecular weight sulphated polysaccharides.

Dextran sulphate (MW 5000 and 8000) and a polysulphated glycosaminoglycan (MW 10,000), at concentrations that provided complete protection in a homologous infection assay, failed to block syncytium formation and the resulting cytopathic effect when MT-2 cells were mixed with H9/HIV-1 cells. These substances also had no antiviral activity when added to cells, after virus challenge, at a time when binding and entry were complete. However, a high molecular weight (500,000) dextran sulphate blocked HIV-1 infection at both stages. Thus, the gp120-CD4 interactions mediating HIV-1 binding and HIV-1-induced syncytium formation are differentially affected by this class of polyanionic substances. Furthermore, size may be a determining factor in their potential application as anti-HIV treatment.

Application of an objective biological assay of human interferons to clinical specimens and a survey of a normal population.

A rapid, quantitative, and nonsubjective method of interferon assay is described, which can be readily applied to clinical specimens. Automated data acquisition and data reduction allowed a significant increase in volume per unit of time over existing methodologies. Plasma always yielded higher (usually 2:1) interferon values than did serum obtained simultaneously. Ranges of interferon levels in plasma in normal control populations are reported as well as ranges for clinical virology laboratory technicians and patients with terminal malignancies or collagen vascular diseases.